Comparison of two probe preparation methods using long oligonucleotide microarrays.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Oligonucleotide microarrays are increasingly used for gene expression analysis.
• Limited data exist on the concordance and efficacy of different probe preparation methods for long oligonucleotide arrays.

Purpose of the Study:

• To compare the performance of two labeling methods for oligonucleotide microarrays: Atlas PowerScript fluorescent cDNA (random priming) and T7 in vitro transcription cRNA [poly(T) priming].
• To assess the reproducibility and accuracy of each method and their concordance when used together.

Main Methods:

• Two labeling kits were used: Atlas PowerScript fluorescent cDNA (random priming) and T7 in vitro transcription cRNA [poly(T) priming].
• Microarray experiments were performed using these labeling methods.
• Reproducibility was assessed using correlation coefficients.
• Confirmatory analysis was done using real-time reverse transcription PCR (RT-PCR) for 18 selected genes.

Main Results:

• Both random priming and poly(T) priming showed high intra-assay reproducibility (correlation coefficients of 0.93 and 0.94, respectively).
• A poor correlation was observed when comparing the results from the two labeling methods.
• Real-time RT-PCR validation favored the cDNA fluorescent labeling method over the T7 labeling method.

Conclusions:

• While both methods demonstrate high reproducibility within themselves, their results are not directly comparable.
• Fluorescent cDNA labeling (random priming) appears superior to T7 cRNA labeling (poly(T) priming) for oligonucleotide microarrays.
• Microarray results from poly(T) priming should be interpreted with caution, even with high reproducibility.